Tissue Container for Molecular and Histology Diagnostics Incorporating a Breakable Membrane

ABSTRACT

A container for storing a biological sample for molecular diagnostic testing and/or histological testing is provided. The container includes a first chamber for receiving a sample holder therein, a second chamber, and a closure for enclosing the container. A breakable membrane, such as a piercable foil, extends within the container and separates the two chambers. When the breakable membrane is broken, fluid can pass between the first and second chambers. The membrane may be broken through an activator on the closure, such as a depressible member or a rotatable carrier, causing the sample holder to break through the membrane.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/257,134, filed Oct. 23, 2008, entitled “Tissue Container forMolecular and Histology Diagnostics Incorporating a Breakable Membrane”,which claims priority to U.S. Provisional Patent Application No.60/982,057, filed Oct. 23, 2007, entitled “Tissue Container ForMolecular and Histology Diagnostics Incorporating a Breakable Membrane”,the entire disclosures of each of which are herein incorporated byreference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a tissue sample container. Moreparticularly, the present invention relates to a sample container forcontaining a biological tissue specimen for molecular diagnostic testingand/or histological testing.

Description of Related Art

Biological samples are often obtained by a researcher or clinician fordiagnostic evaluation to determine the presence of certain diseases andto determine an appropriate treatment for the disease. Tissue samplesare often obtained from a patient for molecular diagnostic and nucleicacid analysis, particularly RNA and DNA analysis, which have becomecommon place in research for the treatment of numerous diseases. Anessential requirement for accurate RNA and DNA analysis is the presenceof high quality and intact RNA and DNA within the biological sample.

Oftentimes, the histologic or cytologic analysis will be performedimmediately after the sample is removed from the patient or source toavoid molecular changes that may occur during storage. These changes,such as gene transcription, result from the degradation of the nucleicacids within the sample caused by exposure of an untreated sample tocertain environmental stresses. However, analysis of the sampleimmediately after the sample is collected is often impossible orimpractical. Therefore, it is necessary to provide a system for storinga sample under controlled conditions for a certain period of time whilemaintaining the structural and molecular integrity of the sample.

Traditionally, one way of accomplishing this storage is by submergingthe sample in a single fixative reagent. A typical fixative reagent isten percent (10%) formalin but may also include water, misciblealcohols, ethanol/acetone mixtures, and ethanol/acetic acid mixtures.The containers used for such storage are generally composed of a singleintegral cavity which could house an effective volume of reagent totreat a particular biological tissue sample. The biological tissuesample is placed in the container along with the reagent, the containeris closed, and the sample is then stored and transported while beingpreserved by the fixative agent. An example of such a container can beseen in U.S. Pat. No. 7,147,826 to Haywood et al. Such containers haveexperienced some success in the industry, but are subject to certainlimitations.

SUMMARY OF THE INVENTION

In accordance with an embodiment of the present invention, a containerfor storing a biological sample includes a housing extending between afirst open end and a second end defining a container interior. Thecontainer includes a removable closure for enclosing the first open end,and at least one breakable membrane separating the container interiorinto at least a first chamber and a second chamber. The second chamberis in fluid isolation from the first chamber, with the first chamberaligned with the first open end of the housing and adapted to receive asample holder therein. The breakable membrane is breakable to establishfluid communication between the first chamber and the second chamber.

The sample holder may be detachably connected to the closure forinsertion into the first chamber of the container interior. The closuremay be threadably matable with the housing. In one configuration, thesample holder may be rotatable with respect to the closure to maintainthe sample holder in a substantially stationary position within thefirst chamber during engagement of the closure with the housing.Optionally, the container may include a platform attached to the closureand adapted for receiving the sample holder for insertion into the firstchamber of the container interior. In a further configuration, theclosure may be threadably matable with the housing, and the platform maybe rotatable with respect to the closure to maintain the sample holderin a stationary position within the first chamber during engagement ofthe closure with the housing.

The sample holder may include a closable housing defining an internalcavity for holding a biological sample, with the housing having aplurality of fluid openings adapted for allowing fluid contained withinat least one of the first chamber and the second chamber to pass intothe internal cavity. In one configuration, the sample holder is ahistology cassette. Optionally, a first fluid may be disposed within thefirst chamber and a second fluid may be disposed within the secondchamber with the first fluid being different than the second fluid.

At least one breakable membrane may be a pierceable foil. In a furtherconfiguration, the housing may have a longitudinal axis and thebreakable membrane may extend across at least a portion of the containerinterior transverse to the longitudinal axis, thereby establishing thefirst and second chambers. Alternatively, the housing may have alongitudinal axis, and the breakable membrane may extend across at leasta portion of the container interior parallel to the longitudinal axis,thereby establishing the first and second chambers. The container mayfurther include a second breakable membrane spaced apart from thebreakable membrane and extending across at least a portion of thecontainer interior parallel to the longitudinal axis with the firstchamber being established between the breakable membrane and the secondbreakable membrane.

The container may also include a movable structure extending from thecontainer interior to an exterior of the container such that movement ofthe movable structure causes the breakable membrane to break, therebyestablishing fluid communication between the first and second chambers.The moveable structure may include a depressible element and the sampleholder may be connected to the depressible element such that depressingthe depressible element causes at least a portion of the sample holderto break the breakable membrane. The depressible element may be aflexible elastomeric button. A removable cover may be disposed over thedepressible element to prevent movement of the depressible element.Alternatively, the moveable structure may include a rotatable carrierand the sample holder may be connected to the rotatable carrier suchthat rotation of the rotatable carrier causes at least a portion of thesample holder to break the breakable membrane.

In accordance with another embodiment of the present invention, acontainer for storing a biological sample includes a housing having afirst open end, a second end, and defining a container interior. Thehousing has a longitudinal axis. The container also includes a breakablemembrane extending across the container interior transverse to thelongitudinal axis separating the container interior into at least afirst chamber and a second chamber. The breakable membrane is breakableto establish fluid communication between the first chamber and thesecond chamber. The first chamber is aligned with the first open end ofthe housing and is adapted to receive a sample holder therein. Thecontainer also includes a removable closure for enclosing the first openend. The removable closure includes a depressible element with thesample holder connected therewith, such that depressing the depressibleelement causes at least a portion of the sample holder to break thebreakable membrane.

In accordance with another embodiment of the present invention, acontainer for storing a biological sample includes a housing having afirst open end, a second end, and a sidewall extending therebetweendefining a container interior. The housing has a longitudinal axis. Thecontainer also includes a first breakable membrane extending across thecontainer interior parallel to the longitudinal axis, and a secondbreakable membrane spaced apart from the first breakable membrane andextending across the container interior parallel to the longitudinalaxis. A first chamber is established between the first breakablemembrane and the second breakable membrane, and a second chamber isestablished between the housing wall and at least one of the firstbreakable membrane and the second breakable membrane. The first chamberis aligned with the first open end of the housing and is adapted toreceive a sample holder therein. At least one of the first breakablemembrane and the second breakable membrane is breakable so as toestablish fluid communication between the first and second chambers. Thecontainer further includes a removable closure for enclosing the firstopen end. The removable closure includes a rotatable carrier and havingthe sample holder connected therewith, wherein rotation of the rotatablecarrier causes at least a portion of the sample holder to break at leastone of the first and second the breakable membranes.

In accordance with yet another embodiment of the present invention, amethod of storing a biological sample within at least one liquidincludes the step of providing a container having a housing extendingbetween a first open end and a second end and defining a containerinterior. The container includes at least one breakable membraneseparating the container interior into at least a first chamber and asecond chamber. The first chamber is aligned with the first open end ofthe housing and is adapted to receive a sample holder therein. At leastthe second chamber contains a liquid therein and is isolated from thefirst chamber by the breakable membrane. The method further includes thestep of inserting a sample holder containing a biological sample intothe first chamber of the container housing. The method also includes thestep of breaking the breakable membrane to establish fluid communicationbetween the first chamber and the second chamber, such that the liquidcontained within the second chamber contacts the biological samplewithin the first chamber.

Optionally, the container includes a removable closure for covering thefirst open end of the housing, with the sample holder movably connectedwith the closure. The step of breaking the breakable membrane furtherincludes moving at least a portion of the sample holder with respect tothe closure to break the breakable membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a container for storing a biologicalsample in accordance with an embodiment of the present invention.

FIG. 2 is an exploded perspective view of the container of FIG. 1.

FIG. 3A is a perspective view of a sample holder of the container ofFIG. 1 in an embodiment of the present invention.

FIG. 3B is a perspective view of the sample holder of FIG. 3A shown inan open position.

FIG. 4A is a top perspective view of a closure of the container of FIG.1 in an embodiment of the present invention.

FIG. 4B is a bottom perspective view of the closure of FIG. 4A includinga sample holder therewith.

FIG. 4C is a bottom perspective view of the closure of FIG. 4A includinga sample holder shown separately.

FIG. 5 is a top perspective view of the container housing of thecontainer of FIG. 1 in an embodiment of the present invention.

FIG. 6A is a cross-sectional view of the container taken along lines 6-6of FIG. 1 with the sample holder contained within the first chamber.

FIG. 6B is a cross-sectional view of the container as shown in FIG. 6Aafter the breakable membrane has been broken.

FIG. 6C is a cross-sectional view of a container shown in an alternateembodiment with the breakable membrane extending across cover 28.

FIG. 7 is a perspective view of a container system for storing abiological sample in accordance with a further embodiment of the presentinvention.

FIG. 8 is an exploded perspective view of the housing of the containerof FIG. 8.

FIG. 9 is an exploded perspective view of the container of FIG. 7.

FIG. 10 is a top perspective view of a closure and housing of thecontainer of FIG. 7 in an embodiment of the present invention, includinga sample holder.

FIG. 11 is a cross sectional view of the container taken along lines11-11 of FIG. 7.

FIG. 12 is a cross sectional view of the container taken along lines12-12 of FIG. 7.

FIG. 13A is a cross sectional view of the container taken along lines13-13 of FIG. 12 shown with the membrane intact.

FIG. 13B is a cross sectional view of the container as in FIG. 13A shownwith the membrane broken.

FIG. 14A is a perspective view of an alternative embodiment of aplatform for use in connection with the present invention.

FIG. 14B is a front view of the platform of FIG. 14A.

FIG. 14C is a side sectional view of the platform taken along line A-Aof FIG. 14B.

FIG. 14D is a side view of the platform of FIG. 14A.

FIG. 14E is a top view of the platform of FIG. 14A.

DESCRIPTION OF PREFERRED EMBODIMENTS

For purposes of the description hereinafter, spatial orientation terms,if used, shall relate to the referenced embodiment as it is oriented inthe accompanying drawing figures or otherwise described in the followingdetailed description. However, it is to be understood that theembodiments described hereinafter may assume many alternative variationsand embodiments. It is also to be understood that the specific devicesillustrated in the accompanying drawing figures and described herein aresimply exemplary and should not be considered as limiting.

The container of the present invention allows for storage of abiological sample, such as a tissue sample for molecular and histologydiagnostics, and in particular histopathology testing. In particular,the container includes an open end and a closed end, with a breakablemembrane separating the container interior into a first chamber and asecond chamber in fluid isolation from each other. Accordingly, a liquidmedium may be contained in at least one of the chambers, such as thesecond chamber. In this manner, a tissue sample contained in, forexample, the first chamber may be handled or processed prior tocontacting the tissue with the solution in the second chamber. As willbe discussed in greater detail herein, in one embodiment of theinvention, the first chamber may be empty representing a storagechamber, and the second chamber may include a liquid medium, such as areagent in the form of a tissue fixative solution for fixing a samplefor histopathology diagnostics. In this manner, a tissue sample may beplaced within the first chamber, and when desired, the membraneseparating the first and second chambers may be broken so as to placethe tissue sample in fluid contact with the solution within the secondchamber.

In a further embodiment of the invention, the first chamber may containa first fluid, such as a tissue fixative solution, and the secondchamber may contain a second fluid, such as a reagent in the form of anucleic acid stabilization solution, such that a tissue sample may beplaced in the first chamber in fluid contact with the first fluid for adesired time period, after which the membrane separating the firstchamber from the second chamber may be broken so as to also place thetissue sample in fluid contact with the solution within the secondchamber. The embodiments described herein are representative ofcontainers capable of use in any of these manners.

Referring to the drawings, in which like reference characters refer tothe like parts throughout the several views thereof, FIGS. 1-6illustrate a container 10 in accordance with an embodiment of thepresent invention. Generally, container 10 includes a housing 12, afirst chamber 20, a second chamber 26, a breakable membrane 32, aclosure 50, and a sample holder 40. The individual components ofcontainer 10 may be made of any suitable material that is impervious toliquid and/or gas, such as glass and/or plastic. In one embodiment, thehousing 12 may be made of one or more than one of the followingrepresentative materials: polypropylene, polyethylene terephthalate(PET), glass, or combinations thereof.

Container 10 generally includes a housing 12 having a housing wall 14extending between a first open end 16 and a second end 18, defining anaxis X of the container, designated for purposes herein as a central orlongitudinal axis, along with a container interior. As will be discussedin more detail herein, a breakable membrane 32 extends transverselyacross the axis of the interior of housing 12 separating housing 12 intoa first chamber 20 and a second chamber 26.

In particular, housing wall 14 defines first chamber 20, with first openend 16 extending into the first chamber 20. First chamber 20 defines afirst intended fill volume and may include a cavity that may be sized soas to receive and accommodate sample holder 40 therein, as will bediscussed in more detail. For example, first chamber 20 may include abottom wall surface defined by breakable membrane 32. Breakable membrane32 may extend entirely across the interior of housing 12, therebyextending about the entire interior perimeter or surface of housing wall14. Alternatively, a wall surface 22 may extend radially inwardly fromportions of the interior of housing wall 14 across the interior ofhousing 12, with downwardly extending side wall surfaces 24 a, 24 b, 24c, and 24 d and the bottom wall surface defined by breakable membrane 32defining a generally rectangular shaped cavity generally correspondingto the size and shape of sample holder 40.

Housing wall 14 further defines second chamber 26 below breakablemembrane 32, defining a second intended fill volume, which is desirablydifferent than the first intended fill volume of the first chamber 20.Second chamber 26 may be positioned adjacent the bottom or second end 18of housing 12. Second end 18 may be an open end extending into secondchamber 26. In such an arrangement, container 10 further includes acover 28 for mating with housing 12 over the second end 18, therebyproviding a closable access to second chamber 26. Cover 28 may bematable with housing 12 in any manner, such as a frictional fit, snapfit, threadable engagement, interlocking structural engagement, or othermanner, providing a liquid tight seal. For example, correspondingthreads may be provided about the perimeter of an external surface ofcover 28 and within the perimeter of an internal surface of housing wall14 of housing 12 at second end 18, or may be provided within theperimeter of an internal surface of cover 28 and about the perimeter ofan external surface of housing wall 14 of housing 12 at second end 18.

In an alternative embodiment shown in FIG. 6C, breakable membrane 32 amay extend across cover 28 as opposed to extending between surfaces 24a, 24 b, 24 c, and 24 d. In this manner, second chamber 26 is formedbelow breakable membrane 32 a as a separate structure with cover 28,which can be matable with housing 12 as described above.

Sample holder 40 is further provided for use with container 10 and isadapted to be received within first chamber 20 of housing 12. Sampleholder 40 may form a part of container 10 or may be separately providedfor use with container 10. Sample holder 40 may be in the form of aconventional histology cassette (a “histo-cassette”) as is known in theart for storing a biological tissue sample during preparation of thesample for diagnostic testing. Such sample holders or histo-cassettesare known for containing biological specimens during processing withfluids to prepare the specimen for later analyses. Typically, suchsample holders or histo-cassettes are generally rectangular, planarhousing structures having an internal cavity, with a plurality ofopenings through the wall surface to provide fluid flow through thehousing. Often, a removable or openable cover encloses the structure,such as through a hinge situated along one end of the housing structurefor providing a door-like cover to the housing structure. Also, a planarsurface, which may be slanted, is often provided in such sample holdersor histo-cassettes acting as a surface for labeling or writing. Thedimensions for such a sample holder, for example, may include a heightof about 0.3 inch (plus or minus 0.1 inch), a length of about 1.73inches (plus or minus 0.1 inch), and a width of about 1.12 inches (plusor minus 0.1 inch). Examples of sample holders that may be useful hereinare shown in U.S. Pat. No. 4,220,252 to Beall et al. and U.S. Pat. No.4,034,884 to White, both of which are expressly incorporated herein byreference.

For example, as shown in FIGS. 3A and 3B, sample holder 40 includes agenerally rectangular planar housing 42 having opposing walls definingan internal cavity 44 for holding a biological tissue sample therein. Atleast one of the walls of housing 42 may be slanted, such as slantedwall 45, providing a surface for applying a label or for writing, so asto provide a mechanism for identification of a sample contained withinsample holder 40, as appropriate. Housing 42 of sample holder 40 is aclosable structure, and may include a hinged door-like structure 46attached with housing 42 thereby permitting access to the internalcavity 44 for storing a tissue sample within or removing a tissue samplefrom internal cavity 44. The door-like structure 46 may be integrallyformed with housing 42 so as to provide a unitary structure with thedoor 46 connected to housing 42 through a flap to provide a mechanismfor pivoting door 46 with respect to housing 42, or door 46 may beotherwise connectable to housing 42, such as through a pivot point 43acting as a hinge for opening door 46 from one side of housing 42 togain access to the internal cavity 44. Housing 42 of sample holder 40includes at least one, and preferably a plurality of, fluid openings 48adapted to allow fluid to flow therethrough. In this manner, whenhousing 42 is positioned within first chamber 20, fluid within firstchamber 20 can flow through openings 48 and contact the biologicaltissue sample contained within internal cavity 44.

Container 10 further includes closure 50 for enclosing the first openend 16 of housing 12. Closure 50 is matable with housing 12 at firstopen end 16 in any manner, such as a frictional fit, snap fit,threadable engagement, interlocking structural engagement, or othermanner providing a liquid tight seal. Desirably, closure 50 and housing12 include corresponding threads such that closure 50 can be threadedwith housing 12 to provide a liquid tight seal therebetween. Forexample, such corresponding threads may be provided about the perimeterof an external surface of closure 50 and within the perimeter of aninternal surface of housing wall 14 of housing 12 at first end 16 or maybe provided within the perimeter of an internal surface of closure 50and about the perimeter of an external surface of housing wall 14 ofhousing 12 at first end 16.

As noted, sample holder 40 may be provided as a separate element for usewithin first chamber 20, or may be interconnected with a part ofcontainer 10. Desirably, sample holder 40 is mated with closure 50. Suchmating may be accomplished by providing sample holder 40 as an integralpart connected to or formed with closure 50 or sample holder 40 may be aseparate structure that is removably matable or detachably connectedwith closure 50. As shown in FIG. 4C, closure 50 may include a platform52 extending from a bottom surface of closure 50 for accommodatingsample holder 40 therein. Platform 52 may include structure formaintaining sample holder 40 attached to closure 50 as shown in FIG. 4B,such as in a snap-fit engagement, and sample holder 40 may be releasablefrom platform 52. In particular, platform 52 may be a generallyrectangular structure defining a rectangular recess for accommodatingthe general size and shape of sample holder 40. Platform 52 may includeone or more fingers 62 extending therefrom for engaging with sampleholder 40, thereby maintaining sample holder 40 within the recessdefined by platform 52. Such fingers 62 may be deflectable, such thatwhen an edge of sample holder 40 adjacent the slanted wall 45 is held inplace against a corresponding finger or protrusion 65 of the platform 52and sample holder 40 is pushed into the recess of platform 52, fingers62 deflect away from the walls of sample holder 40 and then return totheir initial position against nubs 64 of holder 40, thereby snappingsample holder 40 in place. Fingers 62 may lock sample holder 40 in placepermanently with respect to platform 52 and closure 50 or may bedeflectable so as to remove sample holder 40 from platform 52 ifdesired.

Platform 52 may also be provided with a general shape so as to permitopening of door 46 of sample holder 40 while maintaining housing 42 ofsample holder 40 contained therein, thereby providing access to theinterior cavity 44 of sample holder 40 while sample holder 40 is held inplace within platform 52 and with respect to closure 50. For example,the wall surface of platform 52 may have a cut-away portion 57 toaccommodating a handle-like protrustion 47 of door 46, and the overalldimensions and height of the walls of platform 52 may be designed so asto provide for manually opening of the door 46 by contact of handle 47and pivoting of door 46 across platform 52 without interference. Thewalls of platform 52 may further include puncturing elements in the formof piercing points 80, which are oriented in a direction towardbreakable membrane 32 and designed to as to pierce or tear throughbreakable membrane 32 when piercing points 80 are contacted withbreakable membrane 32 such as through application of pressure toplatform 52 against breakable membrane 32, as will be discussed herein.

Closure 50 may further include a depressible element, such as adepressible button 54, which is downwardly deflectable from the closure50 into the interior of first chamber 20 of housing 12. Button 54 isinterconnected with sample holder 40, such as through platform 52. Inparticular, button 54 may include a recessed portion 56 on the interiorthereof for accommodating an extending finger 58 of platform 52, therebymaintaining platform 52 with respect to button 54 and with respect toclosure 50. Button 54 may be constructed of any suitable materialadapted to cause the platform to deflect into the interior of firstchamber 20, such as a flexible or elastomeric polymer.

As noted above, first chamber 20 may be sized so as to receive andaccommodate sample holder 40 therein. In such an arrangement, whensample holder 40 is mated with closure 50 and closure 50 is rotatablyengagement with housing 12, such as through a threaded engagement,sample holder 40 may be provided for rotation with respect to closure50. This may be accomplished, for example, by providing platform 52 as astructure which is rotatable with respect to closure 50, such as througha rotatable connection provided through finger 58 of platform 52extending within a recessed portion 56 of button 54 of closure 50 and byproviding sample holder 40 within platform 52. In this manner, whensample holder 40 is placed within first chamber 20 and closure 50 isrotatably engaged with housing 12, one or both of the platform 52 and/orsample holder 40 will contact one or more of the side wall surfaces 24a, 24 b, 24 c, and 24 d upon rotation of closure 50, thereby maintainingsample holder 40 in place within first chamber 20 of housing 12 ofcontainer 10.

Breakable membrane 32 provides housing 12 with structural features, suchthat first chamber 20 and second chamber 26 may be selectively placed influid communication with each other. For example, with breakablemembrane 32 extending transversely across the housing 12, first chamber20 and second chamber 26 are in fluid isolation, such that any fluidcontained within the first chamber 20 and/or second chamber 26 isisolated from the other chamber. In order to provide fluid communicationbetween the first chamber 20 and the second chamber 26, the breakablemembrane 32 must be broken. Desirably, breakable membrane 32 isconstructed of a material that is easily rupturable or broken open byapplication of pressure thereto. For example, breakable membrane 32 maybe a polymeric material, and is desirably a piercable foil, such asthose commonly used in the packaging industry.

Container 10 may be assembled and provided with liquid media, such assolutions or reagents, stored within first chamber 20 and/or secondchamber 26 at the point of manufacture. Alternatively, any such liquidmedia may be filled into the first chamber 20 and/or the second chamber26 at any point prior to use, such as directly prior to inserting atissue sample into sample holder 40.

As noted, container 10 may be provided for use with a one reagentsystem. In this manner, a single reagent solution, such as a tissuefixative like formalin, may be provided within second chamber 26. Suchfixative solutions stabilize the RNA within a tissue sample forconducting molecular diagnostic testing. Alternatively, container 10 maybe provided for use with a two solution or a two reagent system. Forexample, a wash solution may be provided in second chamber 26, so as todilute the first reagent fixative in the first chamber 20. It is alsopossible that each chamber contains the same reagent since it may beadvantageous to refresh the same reagent after a period of time haspassed. Or, a first reagent solution, such as a tissue fixative likeformalin, may be used within first chamber 20, and a second reagentsolution, such as a stabilizer in the form of a nucleic acidstabilization reagent, for stabilizing the morphology of the tissuesample, may be provided within second chamber 26.

Any reagents may be used with the container of the present invention.For example, the fixative may be formalin, ethanol solutions, Carnoy'ssolution I (ethanol and acetic acid), Carnoy's Solution II (ethanol,chloroform and acetic acid), methacarn (methanol, chloroform and aceticacid), Clark's fixative, Boonfix, and the like. A non-limiting list ofcommercially available fixatives includes, but is not limited to,MIRSKY'S FIXATIVE (available from National Diagnostics, Inc. of Atlanta,Ga.); GLYOFIX (available from Shandon Lipshaw, Inc. of Pittsburgh, Pa.);HISTOCHOICE (available from Amresco); HISTOFIX (available from TrendScientific, New Brighton, Minn.); KRYOFIX (available from Merck);MICROFIX (available from Energy Beam Sciences, Inc., East Granbury,Conn.); NEOFIX (available from Merck); NOTOX (available from Earth SafeIndustries, Inc., Belle Mead, N.J.); OMNIFIX II and OMNIFIX 2000(available from AnCon Genetics, Inc, Mellville, N.Y.); PREFER (availablefrom Anatech Ltd, Battle Creek, Mich.); PRESERVE (available from EnergyBeam Sciences, Inc., East Granbury, Conn.); SAFEFIX II (available fromThermo Fischer Scientific, Inc.); STATFIX (available from StatLabMedical Products, Inc. of Lewisville, Tex.); STF (Streck TissueFixative, available from Streck Laboratories, Omaha, Nebr.); UMFIX(available from Sakura Finetek USA, Inc., Torrance, Calif.); and FINEFIX(available from Milestone Medical of Shelton, Conn.). Commerciallyavailable stabilizers include, but are not limited to, RNALATER(available from Ambion, Inc., Austin Tex.); and RNEASY (available fromQiagen, Inc., Valencia, Calif.). Any other reagents known or hereafterdiscovered for use as fixatives and/or stabilizers are intended asuseful in the present invention.

To assemble container 10, second chamber 26 is filled with the desiredliquid medium. In embodiments where second end 18 is a closed end, suchliquid medium can be supplied within second chamber 26 through a port oropening. Alternatively, housing 12 is provided with an open second end18, with cover 28 placed over second end 18 and mated therewith afterfilling second chamber 26 to contain the liquid medium within secondchamber 26. Thereafter, first chamber 20 may be filled with a differentliquid medium (for example, in embodiments involving a two reagentsystem) through first open end 16. Closure 50, with or without sampleholder 40 extending therefrom, is then placed over the first open end 16of housing 12 and threadably mated therewith. The container 10 thusassembled may be packaged in a separate package, if desired, and storedfor use.

In use, a biological sample, such as a tissue sample extracted from apatient for molecular or histology diagnostics testing, is placed withincavity 44 within sample holder 40, such as through the hinged door 46.In embodiments where sample holder 40 is provided as a separate element,closure 50 can be removed from housing 12 and sample holder 40 may thenbe inserted into the platform 52 of closure 50. Alternatively, if sampleholder 40 is provided with closure 50, the tissue sample may be placedwithin sample holder 40 after closure 50 is removed from housing 12,either with sample holder 40 connected thereto or by removing sampleholder 40 therefrom and then reattaching it thereto.

Closure 50 with sample holder 40 containing the tissue sample therein isthereafter placed over the first open end 16 of housing 12, with sampleholder 40 aligned within and placed into first chamber 20. Closure 50 isthen mated with housing 12, such as by rotating closure 50 and/orhousing 12 with respect to each other in a threaded engagement. Duringsuch respective rotation, sample holder 40 can maintain its orientationwithin first chamber 20 in embodiments in which first chamber 20 issized and oriented for accommodating the particular shape of sampleholder 40 as discussed above.

In embodiments including a one reagent system as discussed above, thetissue sample at this point is contained within sample holder 40 infirst chamber 20 in isolation from the reagent within second chamber 26.When it is desired to contact the tissue sample with the reagent, button54 is depressed, thereby causing the sample holder 40 connectedtherewith to move downwardly and contact breakable membrane 32. Suchpressure causes breakable membrane 32 to break (as shown in FIG. 6B) toestablish fluid communication between first chamber 20 and secondchamber 26. Desirably, platform 52 may include a puncture element, suchas piercing points 80 on platform 52, that cause breakable membrane 32to break when button 54 is depressed. In yet a further embodiment, aseparate element may be provided extending through a portion ofcontainer 10, such as through closure 50 or housing 12, that can bemanipulated so as to cause breaking of breakable membrane 32.

After breakable membrane 32 is broken, container 10 may be inverted,shaken, or otherwise moved so as to cause the reagent within secondchamber 26 to flow across the barrier point of broken membrane 32 andinto first chamber 20, thereby flowing through the fluid openings ofsample holder 40 to contact the tissue sample contained within cavity 44therein. By maintaining the tissue sample separated from the reagentcontained within the second chamber 26 in this manner, contact betweenthe sample and the reagent can be precisely regulated until a desiredtime, and the length of time of contact of the tissue sample and thereagent can be precisely regulated and monitored.

It is further contemplated that a one reagent system can be used whereinthe reagent is placed within the first chamber 20 and the tissue sampleis immediately contacted with the reagent when placed within the firstchamber 20, and after contact for a desired time period, breakablemembrane 32 may be broken as noted above, so as to drain the reagentfrom the first chamber 20 into the second chamber 26, thereby isolatingthe tissue sample from further contact with the reagent.

In embodiments including a two reagent system as discussed above, whenthe sample holder 40 is placed within first chamber 20, the tissuesample is placed in contact with the first reagent contained withinfirst chamber 20, with such reagent flowing through the fluid openings48 of sample holder 40, thereby contacting the tissue sample containedwithin the internal cavity 44 thereof. The tissue sample can bemaintained in contact with the reagent within the first chamber 20 for aspecified time period, after which time the breakable membrane 32 may bebroken so as to cause fluid flow between the first chamber 20 and thesecond chamber 26. Thus, the second reagent maintained within secondchamber 26 can flow into first chamber 20, thereby contacting the tissuesample contained therein. Moreover, it is contemplated that the firstreagent within the first chamber 20 will likewise flow into the secondchamber 26, thereby mixing with the second reagent. Accordingly, theconcentrations of the first and second reagents can be specificallytailored so as to ensure that any mixing of the two reagents will nothave a deleterious effect on the intended functionality of the reagentwhen contacted with the tissue sample. After the second reagent isdisplaced into first chamber 20 and contacted with the tissue sample fora desired time period, the closure 50 may be removed so as to remove thetissue sample from sample holder 40 for any desired diagnostic testing.

Since sample holder 40 is connected with closure 50, access to thetissue sample contained within sample holder 40 can be achieved byremoving closure 50 from container 10 and inverting it, placing theouter surface on a counter, thereby providing sample holder 40 exposed.Any fluid that is contained within sample holder 40 can drip downwardwithin the bottom or internal surface of closure 50 and be caught by therim surrounding closure 50, thereby preventing any leakage or spillageonto the counter surface. The hinged door 46 of sample holder 40 may beopenable with the sample holder 40 connected with the closure 50,thereby providing a simple access to the tissue sample contained thereinand providing a proper support for maintaining the sample holder 40 inplace without having to physically contact any portion of the sampleholder to hold it in place while accessing the sample, therebypreventing any potential for contamination of the sample based oncontact by the user.

Thereafter, the container 10 may be washed and re-used, or morepreferably, will be discarded to prevent cross-contamination with othersamples.

In a further embodiment shown in FIGS. 7-13B, container 110 includessimilar components as the container 10 described above in connectionwith the embodiment of FIGS. 1-6, but including a rotatable memberintegrated with the closure as opposed to a depressible member forbreaking of the breakable membrane 132. In particular, container 110includes a housing 112, a first chamber 120, a second chamber 126, atleast one, and desirably a pair of breakable membranes 132 and 133, aclosure 150 with a sample holder 140, and a rotatable member 160. Aswith the above described embodiment, the individual components ofcontainer 110 may be made of any suitable material that is impervious toliquid and/or gas, such as glass and/or plastic. In one embodiment, thehousing 112 may be made of one or more than one of the followingrepresentative materials: polypropylene, polyethylene terephthalate(PET), glass, or combinations thereof.

Container 110 generally includes a housing 112 having a housing wall 114extending between a first open end 116 and a second end 118, defining anaxis Y of the container, referred to generally herein as the central orlongitudinal axis of the container, along with a container interior. Aswill be discussed in more detail herein, at least one, and desirably apair of breakable membranes 132, 133 extends parallel to the axis Y ofthe container housing 112 separating the container interior into atleast a first chamber 120 and a second chamber 126.

In particular, housing wall 114 defines first chamber 120, with firstopen end 116 extending into the first chamber 120. First chamber 120defines a first intended fill volume, and may include a cavity that maybe sized so as to receive and accommodate sample holder 140 therein.First chamber 120 and second chambers 126 may be established withbreakable membranes 132, 133 extend directly between opposing sides ofhousing wall 114 across the interior of container 112. In one particularembodiment shown in FIG. 8, housing 112 may be configured by arranging ahousing insert 113 within the housing interior formed by housing wall114. Such a housing insert may include a bottom wall surface 122, aswell as side wall surfaces 124 a and 124 b extending from a top plate125. Breakable membranes 132, 133 extend between bottom wall surface 122and side wall surfaces 124 a, 124 b, thereby defining first chamber 120within the container interior as a generally rectangular-shaped cavitygenerally corresponding to the size and shape of sample holder 140. Inthis manner, breakable membranes 132, 133 along with side wall surfaces124 a, 124 b and bottom wall surface 122 form first chamber 120suspended within housing 112, such that second chamber 126 surroundsfirst chamber 120. Housing 112 may further include support walls 115extending across opposing portions of housing wall 114 providing supportstructure for top plate 125 to sit on. Moreover, support walls 115further define the interior volume of housing 112, thereby providing adesired fill volume for second chamber 126 while maintaining a specifiedouter diameter for housing 114.

In particular, housing wall 114 further defines second chamber 126adjacent first chamber 120, separated and isolated therefrom throughbreakable membranes 132, 133. Second chamber 126 defines a secondintended fill volume, which may be different than the first intendedfill volume of the first chamber 120. Second chamber 126 may extend tothe bottom or second end 118 of housing 112. Second end 118 may be anopen end extending into second chamber 126 with container 110 furtherincluding a separate cover (not shown) for mating with housing 112 overthe second end 118 to provide a closable access to second chamber 126,or second end 118 may be a closed end of container housing 112, with anaccess port (not shown) provided for filling the interior of secondchamber 126.

Sample holder 140 is further provided for use with container 110 and isadapted to be received within first chamber 120 of housing 112. Sampleholder 140 is as described above, with like numbers representing likeparts. As previously described, sample holder 140 may form a part ofcontainer 110 or may be separately provided for use with container 110.Desirably, sample holder 140 includes a closable housing 142 defining aninternal cavity 144 for holding a biological tissue sample, with ahinged door-like structure 146, for accessing the internal cavity 144,and with fluid openings 148 adapted to allow fluid to flow therethrough.

Container 110 further includes closure 150 for enclosing the first openend 116 of housing 112. Closure 150 is matable with housing 112 at firstend 116 in any manner, and desirably, in a threaded engagement withhousing 112 to provide a liquid tight seal therebetween as describedabove. Moreover, sample holder 140 may be mated with closure 150, suchas by providing sample holder 140 as an integral part connected to orformed with closure 150 or as a separate structure that is removablymatable or detachably connected with closure 150, such as through aplatform 152 extending from a bottom surface of closure 150, foraccommodating sample holder 140 therein. For example, platform 152 mayinclude structure for maintaining sample holder 140 attached to closure150 as shown in FIG. 10, such as in a snap-fit engagement, and sampleholder 140 may be releasable from platform 152.

As noted above, first chamber 120 may be sized so as to receive andaccommodate sample holder 140 therein. In such an arrangement, whensample holder 140 is mated with closure 150 and closure 150 is rotatablyengaged with housing 112, such as through a threaded engagement, sampleholder 140 may be provided for rotation with respect to closure 150.This may be accomplished, for example, by providing platform 152 as astructure which is rotatable with respect to closure 150, such asthrough a pivoting connection established between arm 158 of platform152 extending through closure 150, such as through bushing 160, andconnected to an external handle 162, and by providing sample holder 140within platform 152. In this manner, when sample holder 140 is placedwithin first chamber 120 and closure 150 is rotatably engaged withhousing 112, platform 152 and sample holder 140 will rotate with respectto closure 150, thereby maintaining sample holder 140 in place withinfirst chamber 120 of housing 112 of container 110.

Closure 150 further includes a mechanism for manually causing rotationof platform 152 and sample holder 140 with respect to closure 150. Inparticular, handle 162 extends through closure 150 and bushing 160, andconnects with platform 152 and/or sample holder 140. As such, handle 162and platform 152 rotate with respect to closure 150. Desirably, theportion of handle 162 that extends externally of container 110 includesfinger engaging surfaces and may include structure to define theorientation of sample holder 140 within container 110. Moreover, handle162 may include a lock so as to prevent rotation of handle 162 withrespect to housing 112 until a desired time. Such lock may be in theform of any interference engagement between the platform 152 and aportion of the housing 112 that prevents rotation of the platform 152 toa position that will cause breakable membranes 132, 133 to rupture untila threshold is overcome.

Breakable membranes 132, 133 provide housing 112 with structuralfeatures such that first chamber 120 and second chamber 126 may beselectively placed in fluid communication with each other. For example,with breakable membranes 132, 133 extending parallel to the axis definedby the first and second ends of housing 112 between bottom wall surface122 and side wall surfaces 124 a and 124 b of housing insert 113, firstchamber 120 and second chamber 126 are in fluid isolation, such that anyfluid contained within the first chamber 120 and/or second chamber 126is isolated from the other chamber. In order to provide fluidcommunication between the first chamber 120 and the second chamber 126,one or both of the breakable membranes 132, 133 must be broken.Desirably, breakable membranes 132, 133 are constructed of a materialthat is easily rupturable or broken open upon application of pressurethereto, such as a piercable foil, as discussed above. Also, platform152 may include piercing points 180 for causing such rupturing ortearing of foil upon contact therewith.

Container 110 may be assembled and provided with liquid media, such assolutions or reagents, stored within first chamber 120 and/or secondchamber 126, either at the point of manufacture or at any point prior touse. Moreover, container 110 may be provided for use with a one reagentsystem or a two reagent system, as described in connection with theembodiment of FIGS. 1-6.

To assemble container 110, second chamber 126 is filled with the desiredliquid medium, such as through an opening or port, or by providing thesecond end 118 as an open end with a separate cover. Alternatively,housing 112 may first be filled with the desired liquid medium throughfirst open end 116 and housing insert 113 including breakable membranes132, 133 contiguous therewith can be inserted into housing 112 throughopen end 116 and secured in position therein. Thereafter, first chamber120 may be filled with a different liquid medium (for example, inembodiments involving a two reagent system) through the open end.Closure 150, with or without sample holder 140 extending therefrom, isthen placed over the first open end 116 of housing 112 and threadablymated therewith, such as by rotating closure 150 with respect to housing112. During such rotation, handle 162 rotates with respect to closure150 through bushing 160, so as to prevent internal rotation of platform152 and sample holder 140, thereby preventing premature breaking ofmembranes 132, 133. The container 110 thus assembled may be packaged ina separate package, if desired, and stored for use.

In use, a biological sample, such as a tissue sample extracted from apatient for molecular or histology diagnostics testing is placed withincavity 144 within sample holder 140, and closure 150 with sample holder140 containing the tissue sample therein is thereafter placed over thefirst open end 116 of housing 112, with sample holder 140 aligned withinand placed into first chamber 120, and closure 150 is then mated withhousing 112. When it is desired to contact the tissue sample with thereagent in second chamber 126, handle 162 is rotated with respect tocontainer housing 112, thereby causing the sample holder 140 connectedtherewith through the platform 152 to move about the axis of container110 and to contact one or both of breakable membranes 132, 133. Suchrotation causes piercing points 180 to contact and tear or break throughone or both of breakable membranes 132, 133 (as shown in FIG. 13B) toestablish fluid communication between first chamber 120 and secondchamber 126. Container 110 may be inverted, shaken, or otherwise movedso as to cause the reagent within second chamber 126 to flow across thebarrier point of broken membranes 132, 133 and into first chamber 120,thereby contacting the tissue sample contained within sample holder 140therein.

As noted, container 110 may be used in connection with a one reagentsystem or a two reagent system as described in connection with theembodiment of FIGS. 1-6. Accordingly, the description of contact of thesample as discussed above applies similarly to the present embodiment.

In one embodiment, the platform may include structure making it capableof accommodating histo-cassettes or sample holders of different sizesand shapes. For example, as shown in an alternate embodiment depicted inFIGS. 14A-14E, platform 52 a may include fingers 90 a and 92 a, whichact as compressible elements for bearing against the wall surfaces ofsample holders of various sizes. Such fingers 90 a and 92 a may act asbiasing elements or leaf springs for exerting a biasing force againstthe wall surface of a sample holder placed within platform 52 a, biasingthe sample holder against the sidewalls of platform 52 a to hold thesample holder in place. More particularly, fingers 90 a apply a biasingforce against a sample holder contained within platform 52 a, whileopposing surface 91 a holds an end of the sample holder therein andfinger or protrusion 65 a holds a separate edge of the sample holdertherein. Also, finger 92 a applies a biasing force against the sampleholder while opposing protrusion 65 a holds the end of the sample holderin place. Such opposite and equal forces assist in maintaining sampleholders of various sizes and shapes in place. Further, wall cut-awayportion 57 a may also be provided for accommodating a handle portion ofthe door of the sample holder, as discussed above, while also providingaccess to the handle portion for opening of the door while the sampleholder is in place in the platform, if desired. In this manner,container 10 may be provided with a single platform that can accommodatevarious sizes and shapes of histo-cassettes therein for use withcontainer 10. Additionally, platform 52 a may include a plurality ofholes 98 a for fluid flow therethrough, as discussed above. Such holes98 a may include a pattern or orientation such that fluid flow throughthe platform to the sample holder will be sufficient for contact with asample contained within the sample holder regardless of the size, shape,and/or geometry of the sample holder. Such a platform 52 a can be usedin place of the platforms described above in connection with theembodiments of FIGS. 1-6 or FIGS. 7-13.

While embodiments of the present invention are satisfied in manydifferent forms, there is shown in the figures and described herein indetail specific embodiments of the invention, with the understandingthat the present disclosure is to be considered as exemplary of theprinciples of the invention and is not intended to limit the inventionto the embodiments illustrated. Various other embodiments will beapparent to, and readily made by those skilled in the art, withoutdeparting from the scope and spirit of the invention. The scope of theinvention will be measured by the appended claims and their equivalents.

What is claimed is:
 1. A container for storing a biological sample,comprising: a housing having a first open end, a second end, and asidewall extending between the first open end and the second end anddefining a container interior; a removable closure for enclosing thefirst open end; and at least one breakable membrane separating thecontainer interior into at least a first chamber and a second chamber incomplete fluid isolation from the first chamber, wherein the firstchamber is open to the first open end of the housing and is adapted toreceive a sample holder therein, the breakable membrane breakable toestablish fluid communication between the first chamber and the secondchamber.
 2. The container of claim 1, wherein the sample holder isdetachably connected to the closure for insertion into the first chamberof the container interior.
 3. The container of claim 1, wherein theclosure is threadably matable with the housing.
 4. The container ofclaim 1, wherein the sample holder is rotatable with respect to theclosure to maintain the sample holder in a substantially stationaryposition within the first chamber during engagement of the closure withthe housing.
 5. The container of claim 1, further comprising a platformattached to the closure and adapted for receiving the sample holder forinsertion into the first chamber of the container interior.
 6. Thecontainer of claim 5, wherein the closure is threadably matable with thehousing and wherein the platform is rotatable with respect to theclosure to maintain the sample holder in a stationary position withinthe first chamber during engagement of the closure with the housing. 7.The container of claim 1, wherein the sample holder comprises a closablehousing defining an internal cavity for holding a biological sample, thehousing comprising a plurality of fluid openings adapted for allowingfluid contained within at least one of the first chamber and the secondchamber to pass into the internal cavity.
 8. The container of claim 1,wherein the sample holder is a histology cassette.
 9. The container ofclaim 1, further comprising a first fluid disposed within the firstchamber and a second fluid disposed within the second chamber, whereinthe first fluid is different than the second fluid.
 10. The container ofclaim 1, wherein the at least one breakable membrane is a pierceablefoil.
 11. The container of claim 1, wherein the housing furthercomprises a longitudinal axis, and the breakable membrane extends acrossat least a portion of the container interior transverse to thelongitudinal axis, thereby establishing the first and second chambers.12. The container of claim 1, wherein the housing further comprises alongitudinal axis, and the breakable membrane extends across at least aportion of the container interior parallel to the longitudinal axis,thereby establishing the first and second chambers.
 13. The container ofclaim 12, further comprising a second breakable membrane spaced apartfrom the breakable membrane and extending across at least a portion ofthe container interior parallel to the longitudinal axis, wherein thefirst chamber is established between the breakable membrane and thesecond breakable membrane.
 14. The container of claim 1, wherein thecontainer further comprises a movable structure extending from thecontainer interior to an exterior of the container such that movement ofthe movable structure causes the breakable membrane to break, therebyestablishing fluid communication between the first and second chambers.15. The container of claim 14, wherein the moveable structure comprisesa depressible element and wherein the sample holder is connected to thedepressible element such that depressing the depressible element causesat least a portion of the sample holder to break the breakable membrane.16. The container of claim 15, wherein the depressible element comprisesa flexible elastomeric button.
 17. The container of claim 15, furthercomprising a removable cover disposed over the depressible element forpreventing movement of the depressible element.
 18. The container ofclaim 14, wherein the moveable structure comprises a rotatable carrierand wherein the sample holder is connected to the rotatable carrier suchthat rotation of the rotatable carrier causes at least a portion of thesample holder to break the breakable membrane.
 19. A container forstoring a biological sample, comprising: a housing extending between afirst open end and a second end, defining a container interior; at leastone breakable membrane separating the container interior into at least afirst chamber and a second chamber in fluid isolation from the firstchamber, wherein the first chamber is open to the first open end of thehousing and is adapted to receive a sample holder therein, the at leastone breakable membrane breakable to establish fluid communicationbetween the first chamber and the second chamber; and a removableclosure for enclosing the first open end, the removable closure havingan engagement portion that connects the removable closure to the sampleholder, wherein the sample holder is free to rotate with respect to theremovable closure while remaining connected to the removable closure viathe engagement portion.